Case study observational research: inflammatory cytokines in the bronchial epithelial lining fluid of COVID-19 patients with acute hypoxemic respiratory failure.

BACKGROUND: In this study, the concentrations of inflammatory cytokines were measured in the bronchial epithelial lining fluid (ELF) and plasma in patients with acute hypoxemic respiratory failure (AHRF) secondary to severe coronavirus disease 2019 (COVID-19). METHODS: We comprehensively analyzed the concentrations of 25 cytokines in the ELF and plasma of 27 COVID-19 AHRF patients. ELF was collected using the bronchial microsampling method through an endotracheal tube just after patients were intubated for mechanical ventilation. RESULTS: Compared with those in healthy volunteers, the concentrations of interleukin (IL)-6 (median 27.6 pmol/L), IL-8 (1045.1 pmol/L), IL-17A (0.8 pmol/L), IL-25 (1.5 pmol/L), and IL-31 (42.3 pmol/L) were significantly greater in the ELF of COVID-19 patients than in that of volunteers. The concentrations of MCP-1 and MIP-1ß were significantly greater in the plasma of COVID-19 patients than in that of volunteers. The ELF/plasma ratio of IL-8 was the highest among the 25 cytokines, with a median of 737, and the ELF/plasma ratio of IL-6 (median: 218), IL-1ß (202), IL-31 (169), MCP-1 (81), MIP-1ß (55), and TNF-α (47) were lower. CONCLUSIONS: The ELF concentrations of IL-6, IL-8, IL-17A, IL-25, and IL-31 were significantly increased in COVID-19 patients. Although high levels of MIP-1 and MIP-1ß were also detected in the blood samples collected simultaneously with the ELF samples, the results indicated that lung inflammation was highly compartmentalized. Our study demonstrated that a comprehensive analysis of cytokines in the ELF is a feasible approach for understanding lung inflammation and systemic interactions in patients with severe pneumonia.

Prediction of acute lung injury assessed by chest computed tomography, oxygen saturation/fraction of inspired oxygen ratio, and serum lactate dehydrogenase in patients with COVID-19.

INTRODUCTION: In treating acute hypoxemic respiratory failure (AHRF) caused by coronavirus disease 2019 (COVID-19), clinicians choose respiratory therapies such as low-flow nasal cannula oxygenation, high-flow nasal cannula oxygenation, or mechanical ventilation after assessment of the patient's condition. Chest computed tomography (CT) imaging contributes significantly to diagnosing COVID-19 pneumonia. However, the costs and potential harm to patients from radiation exposure need to be considered. This study was performed to predict the quantitative extent of COVID-19 acute lung injury using clinical indicators such as an oxygenation index and blood test results. METHODS: We analyzed data from 192 patients with COVID-19 AHRF. Multiple logistic regression was used to determine correlations between the lung infiltration volume (LIV) and other pathophysiological or biochemical laboratory parameters. RESULTS: Among 13 clinical parameters, we identified the oxygen saturation/fraction of inspired oxygen ratio (SF ratio) and serum lactate dehydrogenase (LD) concentration as factors associated with the LIV. In the binary classification of an LIV of ≥20 % or not and with the borderline LD = 2.2 × [SF ratio]-182.4, the accuracy, precision, diagnostic odds ratio, and area under the summary receiver operating characteristic curve were 0.828, 0.818, 23.400, and 0.870, respectively. CONCLUSIONS: These data suggest that acute lung injury due to COVID-19 pneumonia can be estimated using the SF ratio and LD concentration without a CT scan. These findings may provide significant clinical benefit by allowing clinicians to predict acute lung injury levels using simple, minimally invasive assessment of oxygenation capacity and biochemical blood tests.

SARS-CoV-2 RT-PCR as a universal screening on planned admission in asymptomatic patients.

Universal screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on admission is reportedly beneficial in preventing nosocomial infections. However, some issues remain, including low positivity rate, cost, and time required for testing. We describe SARS-CoV-2 reverse transcription polymerase chain reaction (PCR) for universal screening in asymptomatic patients on planned admissions. In total, 14,574 patients were included between October 12, 2020, and June 23, 2022. The PCR-positive rate for the period was 0.44 % (64/14,574). The PCR positivity for the epidemic period by strain was 0.28 % (95 % confidence interval [CI] 0.12-0.56 %), 0.16 % (95 % CI 0.05-0.37 %), 0.21 % (95 % CI 0.09-0.41 %), and 0.9 % (95 % CI 0.65-1.2 %) for the wild-type strain, Alpha, Delta, and Omicron variants, respectively. The proportion of Ct values < 30 was higher in the first half of the epidemic (first vs. second, 29.4 % [95 % CI 16.9-44.8 %] vs. 16.7 % [95 % CI 6.0-28.5 %]), whereas that of Ct values ≥ 35 increased significantly in the second half (first vs. second, 32.4 % [95 % CI 19.3-47.8 %] vs. 70.0 % [95 % CI 53.5-83.4 %]). Of all positives, 50 % (32/64) had a coronavirus disease (COVID-19) history before PCR screening, with a median of 28 days (10-105) from COVID-19 onset or positive to PCR screening. PCR screening may help detect positives with high viral loads early in the epidemic for each mutant strain, with an increasing proportion of positives with low viral loads later in the epidemic. PCR testing may be unnecessary for recently diagnosed cases and patients in whom reinfection is unlikely.

Serum titer of neutralizing antibodies after COVID-19 vaccination in Japanese patients with inflammatory bowel disease.

Vaccination is an important strategy to reduce the infection rate and adverse events of coronavirus disease 2019 (COVID-19). However, the effect of COVID-19 vaccination for Japanese patients with inflammatory bowel disease (IBD) has not been fully elucidated. In the present study, we investigated the serum titer of neutralizing antibodies after COVID-19 vaccination in patients with IBD, treated with and without immunosuppressive therapy. The study consisted of 108 patients with IBD [76 with ulcerative colitis (UC) and 32 with Crohn's disease (CD)] from the gastroenterology outpatient clinic at the Hospital of the Kyoto Prefectural University of Medicine who underwent anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. The control group included 64 healthy subjects who received the anti-SARS-CoV-2 vaccine. When 10 AU/ml of neutralizing antibodies was used as cut-off value, the positive rates of neutralizing antibodies of patients with UC, patients with DC, and the control group were 97.3%, 84.3%, and 100%, respectively. The neutralizing antibody titer showed no difference between patients treated with and without immunosuppressive therapy. These results indicate that COVID-19 vaccination may be useful in patients with IBD, treated with or without immunosuppressive therapy.

Identification of RCC1-LCK as a novel fusion gene in pediatric erythroid sarcoma.

Erythroid sarcoma is a very rare subtype of myeloid sarcoma with undetermined biological features. Here, we present an infant with a multifocal erythroid sarcoma, diagnosed because the tumor cells were positive for glycophorin A. After acute myeloid leukemia-oriented chemotherapy and surgical resection followed by cord blood transplantation, he has successfully maintained complete remission without any late effects. Total transcriptome analysis of the tumor identified a novel fusion gene, RCC1-LCK, and high LCK expression levels, suggesting that LCK overexpression was involved in leukemogenesis in this case.

[B-cell acute lymphoblastic leukemia with glycophorin A expression].

This study reports a case of a 49-year-old woman having B-cell acute lymphoblastic leukemia with glycophorin A, a representative erythroid marker, expression. According to the WHO criteria for mixed phenotype acute leukemia (MPAL), erythroid lineage is not defined, and to the best of our knowledge, only one other case with erythroid/B-cell biphenotypic acute leukemia has been reported previously. To establish the disease entity and clarify the pathophysiology of erythroid/lymphoid MPAL, additional cases need to be analyzed.

Effectiveness and safety of chronic hepatitis C treatment with direct-acting antivirals in patients with rheumatic diseases: A case-series.

Objectives: To assess the effectiveness and safety of interferon-free direct-acting antiviral (DAA) therapy for patients with concomitant hepatitis C virus (HCV) infection and rheumatic diseases (RDs), including rheumatoid arthritis (RA).Methods: This was a single-center observational case-series study conducted in Japan from 2014 to 2018. The primary endpoint was the sustained virological response (SVR) rate 24 weeks after the end of therapy (EoT24). We also evaluated hepatological and rheumatological outcomes and adverse events.Results: Of the 2314 patients with RDs, 18 received DAA therapy (RA = 11, other RDs = 7). The SVR rate for the initial DAA therapy was 89% (16/18). The remaining two achieved SVR with secondary DAA therapy. Along with HCV elimination, hepatological parameters improved significantly from baseline to EoT24. During the study period, no patients newly developed cirrhosis or HCC after HCV elimination. Several patients showed improvement in RDs activity. In RA patients, the simplified disease activity index decreased significantly from baseline to EoT24 (median [interquartile range]: 11.53 [5.14-14.89] vs. 4.06 [2.08-9.05], respectively). On-treatment adverse events were minimal, while two patients experienced tuberculosis reactivation after EoT.Conclusion: DAA therapy was effective and safe, providing hepatological and rheumatological benefits in HCV-infected patients with RDs. Immune reconstitution following HCV elimination should be noted.

pH balance and lactic acid increase in the vitreous body of diabetes mellitus patients.

Although there have been no previous reports on the pH of the human vitreous body, it has been highly theorized that it changes in patients with diabetes mellitus (DM). In humans, it is necessary to measure the vitreous pH in vitro, which is an important point that presents a major problem, as vitreous pH immediately changes when exposed to air. The purpose of this present study was to report our recent development of an in vitro method for measuring vitreous pH via the combination of 27-gauge (G) vitreous surgery and a blood gas analyzer, as well as our investigative findings on whether or not there is a difference of pH depending on the presence of diabetes mellitus (DM). This cross-sectional study involved 30 subjects [18 subjects without DM (DM-) and 12 subjects with DM (DM+)] with no previous history of ophthalmologic surgery. The DM+ group included 6 cases of proliferative diabetic retinopathy (PDR) and 6 cases of non-PDR (NPDR). The DM- Group was comprised of patients with a macular hole or idiopathic epiretinal membrane. The DM+ Group included patients not only with macular hole or idiopathic epiretinal membrane but also diabetic macular edema, however, patients with obvious vitreous hemorrhage were excluded. In all patients, a vitreous specimen was anaerobically obtained at the start of 27G pars plana vitrectomy, with a venous blood sample being collected immediately prior to surgery. Between the DM- and DM+ subjects, pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), K+, Na+, Ca2+, Cl-, lactate, and glucose were compared. In the items in which a significant difference was found between DM- and DM+, the values between the PDR and NPDR cases were also compared. Our findings showed no significant difference in vitreous and venous-blood pH between the DM- and DM+ subjects. The vitreous biochemical data revealed that Ca2+ significantly reduced and lactate and glucose significantly increased in DM+ compared to DM-. Thus, we compared Ca2+, lactate, and glucose between the PDR and NPDR cases. Although glucose did not significantly change, Ca2+ significantly decreased and lactate significantly increased in the PDR cases. The venous biochemical data revealed that only glucose significantly increased in DM+. The data in all investigated items was found to be significantly different between the vitreous and venous samples. Our findings revealed that lactate increases and Ca2+ decreases in the vitreous body of DM patients, especially those with PDR, probably due to the increased production of lactic acid. However, although the production of lactic acid increased, the pH remained at a nearly constant value, thus suggesting that the human vitreous body has a high buffering capacity.

Utility of Coagulation Markers for the Prediction of Rapidly Progressive Interstitial Lung Disease in Patients with Dermatomyositis.

We aimed to evaluate the utility of coagulation markers for the prediction of rapidly progressive interstitial lung disease (RP-ILD) in patients with dermatomyositis (DM). In this retrospective study, 29 patients with DM-associated ILD were analyzed. The number of patients with RP-ILD was 15 (52%). The baseline clinical and demographic data and laboratory markers were analyzed to identify predictive factors for RP-ILD.The univariate logistic regression analysis demonstrated that in addition to well-known laboratory markers, such as serum ferritin, KL-6, and lymphocyte counts, a prolonged activated partial thromboplastin time (aPTT) ratio at the time of DM-associated ILD diagnosis was useful for predicting RP-ILD. Moreover, the logistic regression model and receiver operating characteristic curve analysis showed that combinations of the aPTT ratio and well-known laboratory markers were significantly effective in predicting RP-ILD. This study suggested that an association between RP-ILD and the coagulation system exists.

Monitoring of minimal residual disease after HLA-mismatched allogeneic hematopoietic stem cell transplantation using six-color based HLA-Flow method in pediatric acute leukemia.

HLA-Flow is a flow cytometry-based method using anti-HLA antibodies against mismatched HLA alleles combined with the antibodies against antigens expressed on leukemic cells. It is a sensitive assay to determine minimal residual disease (MRD) after HLA mismatched hematopoietic stem cell transplantation (HSCT). In this study, we report the results of our HLA-Flow using six-color based multicolor fluorescence-activated cell sorting for HLA-mismatched HSCT. We performed HLA-Flow monitoring after HLA mismatched HSCT from July 2013 to July 2016 in nine patients (three with acute lymphoblastic leukemia, five with acute myeloid leukemia, and one with therapy-related acute myeloid leukemia) for MRD monitoring. We detected a relapse after HSCT in three of the nine patients, two of them at MRD levels. HLA-Flow is a sensitive, fast, and inexpensive method for the detection of MRD in patients with HLA-mismatched HSCT.

Spontaneous Regression of Pulmonary Nodules Presenting as Epstein-Barr Virus-related Atypical Infectious Mononucleosis.

BACKGROUND: Pulmonary nodules associated with Epstein-Barr virus (EBV)-related atypical infectious mononucleosis have rarely been described. OBSERVATIONS: A 12-year-old Japanese boy, upon admission, revealed multiple small round nodules (a total of 7 nodules in 4 to 8 mm size) in the lungs on computed tomography. The hemorrhagic pharyngeal tonsils with hot signals on 18F-fluorodeoxyglucose-positron emission tomography-computed tomography were biopsied revealing the presence of EBV-encoded small nuclear RNA (EBER)-positive cells; however, no lymphoma was noted. The patient was diagnosed as having atypical EBV-infectious mononucleosis associated with primary EBV infection. Pulmonary nodules markedly reduced in numbers and sizes spontaneously over a 2-year period. CONCLUSIONS: Differential diagnosis of pulmonary nodules in childhood should include atypical EBV infection.

[Instruction on Clinical Samples and Ethical Guidelines -How We Should Handle These Matters, Measurements, and Problems in Kyoto Prefectural University Hospital-].

Since 2013, the institutional review board (IRB) of Kyoto Prefectural University of Medicine has adopted an on-line application system for medical research protocols to shorten the term of examination. In the same year, research misconduct regarding valsartan (Diovan®) was disclosed, which prompted our institute to reorganize research ethics especially through the Center for Quality Assurance in Research and Development. Concerning the questions asked by Prof. Tohyama, the answers from the IRB of our institute were as fol- lows: 1) the evaluation of new instruments or reagents should be approved by the IRB if it may be exhibited at an academic meeting or published in a scientific journal, 2) non-invasive non-interventional data collection without prior approval would become a target of verbal warnings by the IRB, and 3) residual samples after laboratory examination could be available for research use if the protocol is explained to the patients with an opt-out guarantee. For research use of residual samples after laboratory examination, we have been able to issue a letter of consent for laboratory examination since 2008. However, unfortunately, this letter does not seem to be convenient for the majority of clinicians in our hospital. A more useful and reasonable method seems neces- sary to assist clinical research using residual samples based on Ethical Guidelines for Medical and Health Research Involving Human Subjects, which was newly published in 2015. [Review].

[Industry-Academia Collaboration in the Clinical Laboratory Field: Chairmen's Introductory Remarks].

Industry-academia collaboration has become essential in contemporary medicine. Therefore, many institutes including university corporations have promoted the establishment of an endowed chair and/or performed collaborative research. This symposium was held to overview the present status of industry-academia collaboration in the clinical laboratory field. As a representative of the industry, Mr. Taniguchi (Sysmex) presented the development process of M2BP Glycosylation Isomer, a new marker for liver fibrosis. Mr. Saitoh (Horiba) introduced the achievements of joint collaborative research with Kyoto Prefectural University of Medicine, especially the practical realization of an automated hematology analyzer capable of simultaneously measuring C-reactive protein. Mr. Setoyama (LSI Medience) presented on the characteristic collaboration between academia and commercial laboratories such as Tsukuba Medical Laboratory of Education and Research (TMER). On the other hand, as a representative of academia, Associate Prof. Imai (Kyoto Prefectural University of Medicine) summarized the necessity of clinical laboratories spread regenerative medicine. Finally, Prof. Koshiba (Hyogo College of Medicine) presented on the industry-academia collaboration in routine laboratory work in his institute.

[Case Conference of Hematological Malignancies Based on the Morphology of Blood Cells: Chairmen's Introductory Remarks].

A case conference of hematological malignancies based on the morphology of blood cells was held as a Joint Symposium of the Japanese Society of Laboratory Medicine and Japanese Society of Laboratory Hematology. This style of joint symposium was held four times from 2012 to 2016, whereas child cases were presented for the first time this year. The 4 cases presented in this symposium were as follows: an infant with Down syn- drome showing an atypical clinical course of transient abnormal myelopoiesis (TAM) and myeloid leukemia, a relatively older girl with juvenile myelomonocytic leukemia (JMML), one adult with acute myeloid leukemia (AML) with inv (16) (p13.1q22); CBFB-MYH11 morphologically resembling AML with t (8;21) (q22;q22.1); RUNX1-RUNX1T1, and one adult with high-grade B-cell lymphoma showing t (14;18) (q32;q21); IGH/BCL2. Each case included pathological and interesting morphological findings that were carefully examined and in- tensively discussed by two experienced commentators and participants, including pediatric hemato- pathologists. The importance of the morphological evaluation of characteristic cells such as immature leukemic blasts or lymphomatous ones was reconfirmed at this conference. In addition, immunological, cytogenetic, and molecular examinations were also essential for the final diagnosis of these cases. [Review].

[Basic Evaluation of Microsemi LC-767CRP as POCT Model for Simultaneous Measurement of CBC and CRP].

Microsemi LC-767CRP (LC-767, Horiba, Ltd.) is capable of simultaneous measuring of complete blood count (CBC) including 3-part differentials (3-part Diff.) of white blood cells (WBC) and C-reactive protein (CRP) in 4 minutes. Data obtained using LC-767 were intra-assay-reproducible (n = 10, CV = 0.6-4.0% for CBC, 0.6-2.5% for 3-part Diff. and 2.8-7.7% for CRP). They also showed the good linearity, no definite carry-over and the excellent correlations with routine instruments in our institution. Concerning CRP, the minimal detectable concentration revealed < 0.1 mg/dL, and prozone was observed in the sample containing > 30 mg/dL of CRP. LC-767 showed better correlation with a routine instrument in monocyte percentage than LC-667, probably due to modification of the hemolysis solution ratio and diluent temperature. In conclusion, LC-767 provided accurate CBC and CRP results, and showed improvement in CRP linearity and monocyte percentage compared with LC-667. LC-767, which is equipped with a bar-code reader with easy accessibility to electronic medical record, is suitable as the next-generation point of care testing model in the era of information and network-oriented medicine.

[Testicular Tumor in a Patient with Delta Thalassemia Complicated with Hereditary Persistence of Fetal Hemoglobin].

A 30s male was diagnosed as having the left testicular tumor in 2010. He received the anti-neoplastic chemotherapy, and could achieve the complete remission. But, he relapsed with solitary retroperitoneal lymph node swelling in 2012, and he was referred to our hospital. Laboratory examination on his admission showed the significant increase of fetal hemoglobin (HbF) up to 16.4%. But, neither anemia nor hemolysis was found at that time. Coexistence of therapy-related myeloid neoplasm or HbF production by metastatic lesion was not definite. Isoelectrofocusing of his hemolysate showed the faint HbA2 in addition to dense HbF band. Molecular analysis of his Hb gene revealed the homozygous (G)gamma-158 (C-T) together with homozygous delta-77(T-C). From these findings, he was diagnosed as having hereditary persistence of HbF (HPFH) and homozygous delta thalassemia. The precise incidence of such combined genetic variation has been unknown because the majority of such cases seem to show no significant clinical symptoms as our case. Whereas, it seems necessary to remind the possibility of such genetic variation when adult patients with various acquired diseases such as testicular tumor or hematologic malignancies show the elevated HbF level.

C/EBPß is involved in the amplification of early granulocyte precursors during candidemia-induced "emergency" granulopoiesis.

Granulopoiesis is tightly regulated to meet host demands during both "steady-state" and "emergency" situations, such as infections. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPß is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1-5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPß was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPß knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPß is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

[Case conference of hematological malignancies based on the morphology of blood cells: chairmen's introductory remarks].

A case conference of hematological malignancies based on the morphology of blood cells was held as a joint symposium of the Japanese Society of Laboratory Medicine and Japanese Society of Laboratory Hematology. Four cases were presented and discussed mainly from the viewpoint of cellular morphology: two cases of acute myeloid leukemia (AML) with myelodysplasia-related changes, one case of adult T-cell leukemia/lymphoma, and one case of chronic myelogenous leukemia in the blast phase. Each case included pathological and morphological findings that were carefully examined and intensively discussed by two experienced commentators and participants. The importance of the morphological evaluation of immature cells such as granular myeloblasts or promyelocytes was reconfirmed at this conference. In addition, immunological, cytogenetic, and molecular examinations were also essential for the final diagnosis of these cases.